Ion channel drug discovery and safety screening are limited by high cost per data point, low throughput, and complexity. To directly address these industry-wide pain points, Librede Inc. is developing an alternative cell-free technology for ion channel screening, based on the measurement of ion channels in artificial droplet membranes. By eliminating cells at the point of measurement, our technology enables much lower running costs and simplified assay development. In preliminary work, we have used our platform to measure TRPM8 and hERG ion channels and obtain drug IC50 and EC50 values in agreement with state-of-the-art ion channel electrophysiology platforms. In Phase I development of our technology, we created an automation- ready prototype array plate, and verified its reliability and high yield by using it to create over 1000 artificial membranes. In the Phase II work proposed here, we will validate our platform with four ion channels: hERG, Kv7.1, TRPV1, and Cav1.2, selected for their relevance to screening and their degree of complexity. We will optimize the methods of preparing these ion channels from commercially available host cells to achieve highly repeatable and reliable conductance measurements. We will then use these preparations to measure the effects of drugs on the channels' conductance in a dose-dependent manner, generating reliable and repeatable IC50 and EC50 values that we will compare to those obtained from automated patch clamp screening instrumentation. These drug measurements will be performed using the array plates we developed in Phase I, allowing us to evaluate their performance for a simulated screening application. During the proposed work, we will collaborate with the research group of Prof. Jacob Schmidt at UCLA who, with the PI, initially developed the proposed technology. The Schmidt group will aid Librede by initially processing and measuring the ion channel preparations and then transferring this technology to Librede to replicate, optimize, scale-up, and ultimately commercialize. At the completion of this work, we will have validated four highly relevant ion channels pharmacologically in our system and demonstrated its operation in assay conditions. This validation will demonstrate our system's performance to potential end users and allow placement of instrument prototypes with identified beta testers and early adopters. The Phase II work proposed here is a critical step in the development of our new cell-free electrophysiology platform which can address the main industry pain points of cost, throughput, and ease of use (Librede's value proposition), and has the potential to significantly impact ion channel drug discovery and safety screening.